PROJECT SUMMARY - PROJECT 3 JCPyV is a small DNA virus that infects a large fraction of the human population. Rarely, in immunosuppressed individuals, JCPyV spreads from kidney epithelial cells into the central nervous system, where it can cause a rapidly fatal demyelination disease, progressive multifocal leukoencephalopathy, PML. The viral and cellular factors responsible for this pathogenic switch are not known, and there are no specific anti-viral agents that inhibit JCPyV. Many aspects of JCPyV capsid disassembly and intracellular trafficking are poorly understood. Recent advances in functional genomics, pseudovirus production and use, and virus/cell biology have shed light on entry of other polyomaviruses, but major inroads have yet to be made in our understanding of JCPyV entry. In this project, we will apply these techniques to identify and characterize cellular proteins that mediate JCPyV entry and intracellular trafficking. This endeavor is made possible by the genetic virologic and cell biology expertise of the DiMaio laboratory, in collaboration with the other project leaders, Drs. Atwood and Stehle. The details of virus entry may underlie the molecular basis for the biological activity of JCPyV during the pathogenesis of PML. Furthermore, study of the complex process of JCPyV entry is likely to reveal potential new anti-viral targets and suggest additional strategies to inhibit JCPyV replication and spread. We will test the requirement for candidate entry factors in JCPyV infection and to characterize the role of these factors with a particular emphasis on developing and deploying new assays to determine where in the virus life cycle the factors act. In addition, in mechanistic studies, we will search for a physical association between the cellular factors and conduct mutational analysis on them to identify their crucial features. Finally, if necessary we will conduct large-scale genetic screens to identify additional cellular genes required for JCPyV infection. These studies will provide mechanistic insight into JCPyV infection and inform the development of new therapeutic approaches. These assays will also be used to characterize entry by the JCPyV mutants generated by Dr. Atwood and Dr. Stehle, and to analyze the effects of viral inhibitors developed by these projects.